U.S. patent application number 12/890225 was filed with the patent office on 2012-03-29 for spatially resolved thermal desorption/ionization coupled with mass spectrometry.
This patent application is currently assigned to UT-Battelle, LLC. Invention is credited to Stephen JESSE, Olga S. OVCHINNIKOVA, Gary J. VAN BERKEL.
Application Number | 20120074306 12/890225 |
Document ID | / |
Family ID | 45869691 |
Filed Date | 2012-03-29 |
United States Patent
Application |
20120074306 |
Kind Code |
A1 |
JESSE; Stephen ; et
al. |
March 29, 2012 |
SPATIALLY RESOLVED THERMAL DESORPTION/IONIZATION COUPLED WITH MASS
SPECTROMETRY
Abstract
A system and method for sub-micron analysis of a chemical
composition of a specimen are described. The method includes
providing a specimen for evaluation and a thermal desorption probe,
thermally desorbing an analyte from a target site of said specimen
using the thermally active tip to form a gaseous analyte, ionizing
the gaseous analyte to form an ionized analyte, and analyzing a
chemical composition of the ionized analyte. The thermally
desorbing step can include heating said thermally active tip to
above 200.degree. C., and positioning the target site and the
thermally active tip such that the heating step forms the gaseous
analyte. The thermal desorption probe can include a thermally
active tip extending from a cantilever body and an apex of the
thermally active tip can have a radius of 250 nm or less;
Inventors: |
JESSE; Stephen; (Knoxville,
TN) ; VAN BERKEL; Gary J.; (Clinton, TN) ;
OVCHINNIKOVA; Olga S.; (Knoxville, TN) |
Assignee: |
UT-Battelle, LLC
Oak Ridge
TN
|
Family ID: |
45869691 |
Appl. No.: |
12/890225 |
Filed: |
September 24, 2010 |
Current U.S.
Class: |
250/282 ;
250/288 |
Current CPC
Class: |
H01J 49/0413 20130101;
G01Q 30/02 20130101; H01J 49/049 20130101; H01J 49/0004 20130101;
G01Q 60/38 20130101 |
Class at
Publication: |
250/282 ;
250/288 |
International
Class: |
H01J 49/00 20060101
H01J049/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0001] This invention was made with government support under
Contract No. DE-AC05-00OR22725 awarded by the U.S. Department of
Energy. The government has certain rights in this invention.
Claims
1. A method of analyzing a chemical composition of a specimen,
comprising: providing a specimen for evaluation and a thermal
desorption probe, having a thermally active tip, wherein an apex of
said thermally active tip has a radius of 250 nm of less; thermally
desorbing an analyte from a target site of said specimen using said
thermally active tip to form a gaseous analyte, wherein said
thermally desorbing step comprises: heating said thermally active
tip to above 200.degree. C., and positioning said target site and
said thermally active tip such that said heating step forms said
gaseous analyte; ionizing said gaseous analyte to form an ionized
analyte; and analyzing a chemical composition of said ionized
analyte.
2. The method according to claim 1, further comprising: determining
a predetermined sampling path comprising a plurality of target
sites prior to said first thermal desorption step, and sequentially
articulating said thermally active tip along said predetermined
sampling path and repeating said thermally desorbing, ionizing and
analyzing steps for each target site.
3. The method according to claim 2, wherein said determining step
comprises analyzing a topography of said specimen utilizing said
thermal desorption probe in an atomic force microscopy mode.
4. The method according to claim 3, wherein said thermal desorption
probe is maintained in contact with said specimen while said
thermal desorption probe is sequentially articulated along said
sampling path.
5. The method according to claim 3, wherein said thermal desorption
probe is intermittently removed from contact with said specimen
between said target sites.
6. The method according to claim 3, wherein said thermal desorption
probe does not contact said specimen during at least one of said
thermal desorbing steps.
7. The method according to claim 3, further comprising: mapping a
property of a chemical component for each of said target sites.
8. The method according to claim 1, further comprising maintaining
said gaseous analyte above a condensation temperature between said
thermal desorption step and said ionizing step.
9. The method according to claim 1, wherein said analyzing step
comprises evaluating said ionized analyte with a mass
spectrometer.
10. The method according to claim 1, wherein said ionizing step
comprises passing said gaseous analyte through an ionization
source.
11. The method according to claim 1, wherein said thermal desorbing
step comprises volatizing said analyte.
12. A system for analyzing a chemical composition of a specimen,
comprising: a specimen stage for supporting a specimen; a thermal
desorption probe, having a thermally active tip, wherein an apex of
said thermally active tip has a radius of 250 nm of less; a
collection device arranged to capture an gaseous analyte desorbed
from a specimen by said thermal desorption probe; a heating device
for maintaining a temperature of a gaseous analyte above a
condensation temperature within said collection device; an
analytical instrument for determining a chemical composition of an
analyte, wherein an outlet of said collection device is coupled to
an inlet of said analytical instrument; and a stepper mechanism
configured to provide relative motion between said specimen stage
and said thermal desorption probe.
13. The system according to claim 12, further comprising a
controller configured for (i) actuating said stepper mechanism to
sequentially articulate said thermal desorption probe and/or said
sample stage along a predetermined sampling path comprising a
plurality of target sites, and (ii) heating said thermally active
tip to a temperature greater than 200.degree. C. while proximate
the target site in order to cause analytes at the plurality of
target sites to form gaseous analytes.
14. The system according to claim 13, wherein said controller is
configured for locally heating said thermally active tip to a
temperature greater than 350.degree. C.
15. The system according to claim 13, wherein said controller is
configured for causing said stepper mechanism to bring said thermal
desorption probe into contact with a specimen at each of said
target sites.
16. The system according to claim 15, herein said controller is
configured for removing said thermal desorption probe from contact
with a specimen while said thermal desorption probe is articulated
along said sampling path.
17. The system according to claim 15, wherein said controller is
configured for articulating said thermal desorption probe along
said sampling path in a noncontact mode.
18. The system according to claim 15, wherein a chemical
composition of an analyte desorbed at each of said plurality of
target sites is determined by said analytical instrument and a plot
of said data is generated.
19. The system according to claim 12, wherein a distance between
said collection device and said thermally active tip is less than
0.1 mm.
20. The system according to claim 12, wherein said analytical
instrument is a mass spectrometer, an ionization source, a
separation method, or a combination thereof.
Description
FIELD OF THE INVENTION
[0002] This invention is drawn to systems and methods for high
spatial-resolution analysis of the chemical composition of a
specimen, in particular, those that include thermal desorption of
an analyte.
BACKGROUND OF THE INVENTION
[0003] Advances in analytical technology have pushed the limit of
human understanding of chemical and physical phenomena. This is
certainly the case in the study of materials and systems in the
nanoscale range. New tools create the opportunity for the new
discoveries. Currently available techniques that allow nanometer,
i.e., sub-micron, resolution evaluations are limited in the amount
of chemical information they can provide. Techniques such as
electron microscopy and scanning probe microscopy (SPM), which
allow for spatial imaging resolution of 1 nm or better, provide
almost no chemical information about the sample. Alternatively,
techniques like RAMAN and IR imaging provide some limited molecular
level chemical information. Mass spectrometry-based techniques that
provide precise molecular mass and chemical structure information
can in some cases provide chemical information at the submicron
level. However, those techniques are currently limited to operation
in high vacuum and often involve highly specialized sample
preparation techniques.
SUMMARY OF THE INVENTION
[0004] A method and system for analyzing a chemical composition of
a specimen is described. The system can include a specimen stage
for supporting a specimen, a thermal desorption probe, a collection
device arranged to capture an gaseous analyte desorbed from a
specimen by the thermal desorption probe, an analytical instrument
for determining a chemical composition of an analyte, and a stepper
mechanism configured to provide relative motion between the
specimen stage and the thermal desorption probe. The thermal
desorption probe can include a thermally active tip extending from
a cantilever body and an apex of the thermally active tip can have
a radius of 250 nm of less. An outlet of the collection device can
be coupled to an inlet of the analytical instrument. In addition,
the system can include a controller configured for (i) actuating
the stepper mechanism to sequentially articulate the thermal
desorption probe and/or the sample stage along a predetermined
sampling path comprising a plurality of target sites, and (ii)
heating the thermally active tip to a temperature greater than
200.degree. C. while proximate the target site in order to cause
analytes at the plurality of target sites to form gaseous
analytes.
[0005] The controller can be configured for causing the stepper
mechanism to bring the thermal desorption probe into contact with a
specimen at each of the target sites. The controller can be
configured for removing the thermal desorption probe from contact
with a specimen while the thermal desorption probe is articulated
along the sampling path. The controller can be controller for
articulating the thermal desorption probe along the sampling path
in a non-contact mode. The system according to claim 12, wherein
said analytical instrument is a mass spectrometer, an ionization
source, a separation method, or a combination thereof.
[0006] The invention also includes a method of analyzing a chemical
composition of a specimen. The method can include providing a
specimen for evaluation and a thermal desorption probe, thermally
desorbing an analyte from a target site of the specimen using the
thermally active tip to form a gaseous analyte, ionizing the
gaseous analyte to form an ionized analyte, and analyzing a
chemical composition of said ionized analyte. The thermally
desorbing step can include heating the thermally active tip to
above 200.degree. C., and positioning the target site and the
thermally active tip such that the heating step forms the gaseous
analyte.
[0007] The method can also include determining a predetermined
sampling path comprising a plurality of target sites prior to the
first thermal desorption step, and sequentially articulating the
thermally active tip along the predetermined sampling path and
repeating the thermally desorbing, ionizing and analyzing steps for
each target site. The determining step can include analyzing a
topography of the specimen utilizing the thermal desorption probe
in an atomic force microscopy mode.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] A fuller understanding of the present invention and the
features and benefits thereof will be obtained upon review of the
following detailed description together with the accompanying
drawings, in which:
[0009] FIG. 1(a) is a schematic of a system according to the
invention, and 1(b) is a close up of FIG. 1(a) showing the gap
between the thermal desorption probe and the collection device.
[0010] FIG. 2 is a cross-sectional view of the system according to
FIG. 1(a) taken along cut line A-A.
[0011] FIG. 3 is a schematic of a thermal desorption probe and a
specimen in non-contact mode where the thermally active tip is
resistance-heated.
[0012] FIG. 4 is a schematic of a thermal desorption probe and a
specimen in contact mode where the thermally active tip is
laser-heated.
[0013] FIG. 5 is a schematic of a thermal desorption probe and a
specimen in contact mode where the thermally active tip is
laser-heated.
[0014] FIG. 6 is a schematic of a system according to the
invention.
[0015] FIG. 7 is a graph showing the calculated diameter of a
desorbed crater as a function of time for a 250 .mu.m diameter
probe tip and a 10 nm diameter probe tip.
[0016] FIG. 8 is a graph showing the calculated number of moles
desorbed by a variety of different diameter thermal probe tips
versus concentration for varying crater diameters.
[0017] FIG. 9(a) is an AFM image of a surface that has been sampled
by a thermal desorption probe system as described herein; FIG. 9(b)
is a graph showing the depth of the craters shown in FIG. 9(a); and
FIG. 9(c) is a chronogram showing the relative intensity of the SRM
caffeine (m/z 195.fwdarw.138) versus time.
[0018] FIG. 10 is a schematic illustration of a thin-layer
chromatography/thermal desorption/ionization-mass spectroscopy
setup used in an example.
[0019] FIG. 11 is charts of normalized SRM intensity for TNT in
APCI negative ion mode versus (a) proximal probe temperature, and
(b) gas flow rate into the ion source of the mass spectrometer.
[0020] FIG. 12 is charts of (a) measured and calculated mass
spectral peak widths, (b) normalized measured peak area, and (c)
normalized peak height versus surface scan speed for Sudan red 7B
using SRM detection in positive ion mode APCI.
[0021] FIG. 13 is charts of normalized SRM peak areas versus amount
spotted on the HPTLC plate for (a) TNT in APCI negative ion mode,
(b) acetaminophen in APCI positive ion mode, and (c) Sudan red 7B
in APCI positive ion mode.
[0022] FIG. 14 is (a) a black and white photograph of glass-hacked
normal-phase silica gel plate development lane showing the
separated bands of caffeine, acetaminophen and aspirin, (b) the
total ion current from full scan ESCi mode, and the individual
extracted ion current chromatograms for (c) caffeine using APCI,
(d) acetaminophen using APCI, and (e) aspirin using ESI.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The present invention is directed to systems and methods for
high spatial-resolution analysis of the chemical composition of a
specimen. In particular, systems capable of achieving sub-micron
resolution utilizing thermal desorption of a specimen. The systems
and methods described herein can also include providing
topographic, mechanical, and chemical mapping of the surface of the
specimen. It is noted that like and corresponding elements
mentioned herein and illustrated in the figures are generally
referred to by the same reference numeral. It is also noted that
proportions of various elements in the accompanying figures are not
drawn to scale to enable clear illustration if elements having
smaller dimensions relative to other elements having larger
dimensions.
[0024] As shown in FIG. 1, the system 10 for analyzing a chemical
composition of a specimen (S) can include a sample stage 12 for
supporting the specimen (S), a thermal desorption probe 14, a
collection device 16, an analytical instrument 18, a stepper
mechanism 20, and a controller 22. The stepper mechanism 20 can be
configured to move the specimen stage 12 and the thermal desorption
probe 14 relative to one another.
[0025] The thermal desorption probe 14 can include a thermally
active tip 24 extending from a cantilever body 26. The thermally
active tip 24 can include a distal that has a conical shape with a
rounded tip. The apex of the thermally active tip 24 can have a
radius of 250 nm or less, or 100 nm or less, or 75 nm or less, or
50 nm or less, or 30 nm or less, or 15 nm or less. The thermally
active tip 24 can be in communication with a heating mechanism such
that the thermally active tip 24 can be heated to a temperature of
at least 200.degree. C., at least 250.degree. C., at least
300.degree. C., at least 350.degree. C., at least 400.degree. C.,
at least 450.degree. C., or at least 500.degree. C..
[0026] The heating mechanism can be a voltage source 28 connected
to a resistive heating circuit coupled to the thermal desorption
probe 14. In such an example, the voltage source can be
electrically coupled to the resistive heating circuit of the
thermal desorption probe 14 and the thermally active tip 24 can be
heated via resistive heating.
[0027] In another example, the heating mechanism can be a laser
beam 30 emitted by a laser 32 and the heating can be facilitated by
directing the laser beam 30 at the thermal desorption probe 14. In
particular, the laser beam 30 can be directed at the thermally
active tip 24 or the cantilever body 26, as shown in FIGS. 4 and 5,
respectively.
[0028] Where the thermally active tip 24 contacts the specimen (S),
the heating mechanism can include frictional force. For example,
the cantilever body 26 can oscillate at a high frequency, e.g., an
ultrasonic frequency, while the thermally active tip 24 contacts
the specimen (S). The repeated contacting of the specimen (S) by
the thermally active tip 24 can provide additional heating or can
be used as an independent method of thermally desorbing the gaseous
analyte 42 from the target site 36.
[0029] The thermal desorption probe 14 can also be designed to
function as an atomic force microscopy probe. Thus, the mechanical
properties of the thermal desorption probe 14 and its components,
the thermally active tip 24 and the cantilever body 26, can be
designed for use for measuring the topography of a surface as part
of an atomic force microscope. The system 10 can include an atomic
force microscopy system 38 for utilizing the thermal desorption
probe 14 for measuring the topography of a surface 40 of the
specimen S.
[0030] During the analysis process, the stepper mechanism 20 can
move the heated thermally active tip 24 and the target site 36 such
that they are proximate to or in contact with one another, as shown
in FIGS. 3 and 4, respectively. This position can be maintained
until a gaseous analyte 42 evolves from the target site 36. The
gaseous analyte 42 can be volatized molecules from the target site
36, pyrolytic decomposition products of molecules from the target
site 36, or both. Generally, desorption of smaller molecules can be
achieved by volatilization, white larger molecules may require
pyrolytic decomposition.
[0031] The collection device 16 of the system 10 can be arranged to
capture a gaseous analyte 42 desorbed from the specimen (S) by the
thermal desorption probe 14. Because of the nanoscale dimensions of
the thermally active tip 24, the quantity of gaseous analyte 42
evolved from an individual target site 36 is miniscule, e.g., on
the order of picomoles or even attomoles.
[0032] Several unique features of the system 10 that enable
analysis of such miniscule quantities are of particular note. For
example, the analysis generally takes place at atmospheric
pressure, rather than in a vacuum as is generally preferred in the
thermal desorption art. In addition, as shown in FIG. 10, the
ionization source 50 and a portion of the collection device 14 are
contained within a heated housing 71. The housing 71 can include
one or more block heaters 74 for maintaining the interior of the
housing 71 at or near the temperature of the thermally active tip
24 when the gaseous analyte 42 was evolved. In addition, the
nebulizing gas tip 72 can be supplied with nebulizing gas that has
been heated by a nebulizing gas heater 76. Thus, the nebulizing gas
can exit the nebulizing gas tip 72 at a temperature at or near the
temperature of the thermally active tip 24 when the gaseous analyte
42 was evolved. For example the block heater 74 and nebulizing gas
heater 76 can heat the relevant gases to temperatures between 100
and 1000.degree. C., or between 150 and 750.degree. C., or between
200 and 500.degree. C., or between 250 and 400.degree. C., or at
least 200.degree. C., or at least 250.degree. C., or at least
300.degree. C., or at least 350.degree. C. The combination of these
features contributes to a highly efficient ionization process that
enables the mass spectrometer to detect the miniscule quantities of
analyte, e.g., attomole quantities, required for nanoscale
resolution using thermal desorption techniques.
[0033] Thus, the methods described herein can include maintaining
said gaseous analyte 42 at a relatively constant temperature as the
gaseous analyte 42 is transported from an inlet 44 to an outlet 46
of the collection device 16. For example, a temperature of the
gaseous analyte 42 can be at least 150.degree. C., at least
200.degree. C., or at least 250.degree. C., or at least 300.degree.
C., or at least 350.degree. C. In particular, the temperature of
the gaseous analyte 42 can be maintained high enough that the
gaseous analyte 42 does not condense on an interior of the
collection device 16.
[0034] An inlet 44 of the collection device 16 can be positioned
proximate the thermally active tip 24. For example, as shown in
FIG. 1(b), a distance (d) between the inlet 44 of the collection
device 16 and the thermally active tip 24 can be 500 .mu.m or less,
or 250 .mu.m or less, or 100 .mu.m or less, or 50 .mu.m or less. In
order to facilitate capture of the gaseous analyte 42, the
collection device inlet 44 can be placed above the thermally active
tip 24, as shown in FIG. 1. Alternately, the collection device
inlet 44 can be placed to the side of the thermally active tip 24,
as shown in FIG. 11.
[0035] An outlet 46 of the collection device 16 can coupled of an
inlet 48 for an analytical instrument 18. An intake flow rate of
the collection device 16 can be between 0.1 mL/min and 60 mL/min,
or between 1 mL/min and 50 ml/min, or between 5 mL/min and 40
mL/min. The pressure at the sample surface can be approximately
atmospheric pressure, e.g., 0.95-1.05 atm.
[0036] A gas outlet 46 of the collection device 16 can be coupled
to a gas inlet 48 of an analytical instrument 18. For example, as
shown in FIG. 1, the collection device 16 can be directly coupled
to the gas inlet 48 of an ionization source 50 and indirectly
coupled to the gas inlet 52 of a mass spectrometer 54. A gas outlet
56 of the ionization source 50 can be directly coupled to the gas
inlet 52 of the mass spectrometer 54. As used herein, where an
inlet and an outlet are coupled, they are physically coupled such
that a gas exiting the outlet is directed into the inlet with
little to no loss to the atmosphere.
[0037] The analytical instrument 18 can be any instrument utilized
for analyzing gaseous analytes. Exemplary analytical instruments
include, but are not limited to, mass spectrometers, ionization
sources, separation methods, and combinations thereof. Exemplary
ionization sources include, but are not limited to electrospray
ionization, atmospheric pressure chemical ionization, atmospheric
pressure photo-ionization or inductively coupled plasma. Exemplary
separation methods include, but are not limited to, atmospheric
pressure ion mobility or differential mobility spectrometery
(post-ionization) and gas chromatography. Exemplary mass
spectrometers ("MS") include, but are not limited to, sector MS,
time-of-flight MS, quadrupole MSS filter MS, three-dimensional
quadrupole ion trap MS, linear quadrupole inn trap MS. Fourier
transform ion cyclotron resonance MS, orbitrap MS and toroidal ion
trap MS. Exemplary ionization sources are electrospray ionization,
atmospheric pressure chemical ionization, and combinations thereof,
i.e., electrospray chemical ionization (ESCi).
[0038] As used herein, a stepper mechanism has its standard meaning
in the art and should be understood to include any device or
combination of devices for changing the relative position between
the thermal desorption probe 14 and the sample stage 12 or the
specimen (S) supported thereon. For example, the sample stage 12
can be coupled to the stepper mechanism 20 and move the sample
stage 12 laterally (X-axis), transversely (Y-axis), and vertically
(Z-axis) along the sampling path 60. Alternately, the thermal
desorption probe 14 can be mounted to the stepper 20, e.g., via the
atomic force microscopy system 38, and can move the thermal
desorption probe 14 laterally, transversely and vertically along
the sampling path 60.
[0039] As shown in FIG. 2, a sampling path 60 can be a sampling
regime that includes a plurality of target sites 36. FIG. 2 only
shows the lateral and transverse component of the sequence for
sampling the target sites 36 along the sampling path 60; however,
the sampling path 60 can also include a vertical component. For
example, as shown in FIG. 10(a), the thermally active tip 24 and a
first target site 36 will be brought into contact for purposes of
thermally desorbing an analyte at the first target site 36, and can
then be separated with the thermally active tip 24 is positioned
above a second target site 36.
[0040] As shown in FIGS. 2 and 10(a), thermal desorption of a
target site will produce a crater 58 from the desorbed molecules.
The diameter of the craters can be 1 .mu.m or less, or 500 nm or
less, or 250 nm or less, or 125 nm or less, or 50 nm or less.
[0041] The thermal desorption can occur with the thermal desorption
probe 14 in contact with the target site 36. The articulation
between sequential target sites 36 can occur with the thermal
desorption probe 14 in contact with the specimen (S) or proximate
to, but not contacting, the specimen (S). Similarly, the thermal
desorption can occur with the thermal desorption probe 14
proximate, but not contacting, the target site 36. The controller
22 can be configured for causing the stepper mechanism 20 to
perform each of the thermal desorption sequences described above,
or anywhere herein.
[0042] In some examples, the target sites 36 can be sampling lines
62. In general, the plurality of sampling lines 62 will be parallel
and spaced apart by a distance (d.sub.s). In such an embodiment,
the specimen (S) can be thermally desorbed along an entire sampling
line 62 and the gaseous analyte 42 analyzed continuously by the
analytical instrument 18. The thermal desorption probe and the
sample stage 12 will then travel along a relocating path 64 prior
to thermally desorbing the next sampling line 62 along the sampling
path 60.
[0043] The sampling path 60 can be an array of regularly spaced
target sites 36. As used herein, "regular spacing" and "regularly
spaced" are used interchangeably and refer to spacing where the
distance between adjacent target sites 36 in a line is equal or
approximately equal along the length of the line, as shown in FIG.
2. Regular spacing also refers to instances where the same target
site is part of two or more lines with regular spacing, which is
also shown in FIG. 2. Of interest, the center-to-center distance
between adjacent target sites 36 can be 5 .mu.m or less, or 3 .mu.m
or less, or 2 .mu.m or less, or 1 .mu.m or less, or 0.5 .mu.m or
less, or 250 nm or less, or 100 nm or less, or 50 nm or less.
[0044] In some instances, the entire sampling path 60 will be
determined prior to beginning the sampling process, i.e., prior to
the thermal desorption of the first target site 36. In such
instances, the surface 40 of the specimen (S) can be scanned to
determine the topography of the specimen (S) using the thermal
desorption probe in atomic force microscopy mode. In such
embodiments, the topography of the specimen (S) can be used to
determine the lateral, transverse and vertical components of the
sampling path 60 prior to thermal desorption of the first target
site 36.
[0045] The data from each of the target sites 36 can be stored in a
computer readable storage, such as are known in the art. The data
can be complied to form a two-dimensional map, or surface, of the
composition of the specimen by plotting the data according to the
array of target sites was obtained. The data can be displayed on an
output device, such as a monitor, printer, smartphone or the
like.
[0046] The system 10 can also include a controller 22 configured
for carrying out any of the method steps described herein. For
example, the controller 22 can be configured for causing the
stepper mechanism 20 to sequentially articulate the thermal
desorption probe 14, the sample stage 12, or both, along a
predetermined sampling path 60 comprising a plurality of target
sites 36. The controller 22 can also be configured for heating the
thermally active tip 14 to a temperature greater than 200.degree.
C. while proximate the target site 36 in order to cause analytes at
the plurality of target sites 36 to form gaseous analytes 34, and
cooling the thermally active tip 14 to approximately room
temperature, e.g., less than 40.degree. C., between thermal
desorption processes.
[0047] The controller 22 can include a computer readable storage 66
in communication with a processor 68. The computer readable storage
66 can include computer executable instructions for carrying out
the methods described herein. The processor 68 can be configured to
execute the computer executable instructions stored on the computer
readable storage 66. The controller 22 can be in communication with
the stepper mechanism 20, the atomic force microscopy system 38,
the analytical instrument 50, the laser 32, the voltage source 28,
the block heater 74 and/or the nebulizing gas heater 76 described
herein. In addition, although shown as a single box that includes a
single computer readable storage 66 and a single processor 68, it
should be understood that the controller 22 can be spread across
multiple devices and can include multiple computer readable
storages and processors.
[0048] As used herein, sequentially articulate refers to
automatically moving the thermal desorption probe 14, the sample
stage 12, or both along the sampling path 60 to a plurality of
target sites 36. In some instances this articulation can be
continuous while in others there will be intermittent pauses. For
example, the articulation may be paused while the target sites 36
are thermally desorbed in order to ensure an adequate amount of
gaseous analyte 42 is evolved from the target site 36, or
articulation may be paused while the thermally active tip 24 is
heated to an adequate temperature for thermal desorption, or to
provide adequate separation between ionized analyte samples being
fed to an analytical instrument 18, such as a mass spectrometer
54.
[0049] A method of analyzing a chemical composition of a specimen
is also described. The method can include providing a specimen (S)
for evaluation and a thermal desorption probe 14. The method can
also include thermally desorbing an analyte from a target site 36
of the specimen (S) using a thermally active tip 24 of the thermal
desorption probe 14 to form a gaseous analyte 42. The thermal
desorption step can include heating the thermally active tip to
above 200.degree. C., and positioning the target site 36 and the
thermally active tip 24 such that the heating step evolves the
gaseous analyte 42. The method can also include ionizing the
gaseous analyte 42 to form an ionized analyte 70 and analyzing a
chemical composition of the ionized analyte 70.
[0050] The method can also include determining a predetermined
sampling path 60 comprising a plurality of target sites 36 prior to
the first thermal desorption step. Additionally, the method can
include sequentially articulating the thermally active tip 24
and/or the sample stage 12 along the predetermined sampling path 60
and repeating the thermally desorbing, ionizing and analyzing steps
for each target site 36. The sequentially articulating step can
include moving the thermally active tip 24, the sample stage 12 or
both, so that the thermally active tip 24 is sequentially
positioned proximate each target site 36 along the sampling path 60
so that each target site can be thermally desorbed.
[0051] The step of determining the predetermined sampling path 60
can include analyzing the topography of the specimen utilizing the
thermal desorption probe 14 in an atomic force microscopy mode.
Analyzing can include mapping the position laterally and
transversely and, optionally, vertically of the target sites 36 and
the sampling path 60.
[0052] The method can also include plotting any exogenous or
endogenous property related to the specimen (S) being evaluated,
including a property of a molecule or chemical component for each
of the target sits 36. Properties of interest include, but are not
limited to, concentration of a molecule or decomposition product,
the relative ratio of two molecules (such as compound and reaction
product of the compound), and the relative ratio of decomposition
products.
[0053] For example, the property of can be the concentration of a
chemical component, such as a pharmaceutical and its metabolites,
at each target site 36. By arranging the data for each target site
spatially within the specimen (S) a two dimensional surface can be
plotted.
[0054] In some exemplary methods, the thermal desorption step can
include volatizing on analyte at the target site 36, pyrolyzing an
analyte at the target site 36, or a combination of both. The
ionizing step can include passing the gaseous analyte 42 through an
ionization source 50 and, independently, the analyzing step can
include evaluating the ionized analyte 70 with a mass spectrometer
54.
EXAMPLES
[0055] The example and calculations provided herein are provided to
demonstrate the ability to achieve nanometer, i.e., sub-micron,
spatial resolution for chemical composition analysis using thermal
desorption techniques. Although the examples described herein are
specific, the potential applications of the coupled AFM/mass
spectrometer system extend far beyond these specific examples. For
example, in the study of polymeric materials for identification of
impurities, or to determine trace amounts of materials locally
isolated to submicron regions in tissues, which can be useful in
the development in pharmaceutical drugs.
Example 1
[0056] Electrospray ionization (ESL) works by ionizing a gaseous
sample through reaction with charged solvent droplets, protonated
solvent species, or gas phase ions created in the ESI process.
Besides the apparent high secondary ionization efficiency, the
other advantage of using ESI is the ability to form
multiply-charged species from macromolecular species.
[0057] In atmospheric pressure chemical ionization (APCI)
ionization occurs by ion/molecule chemistry in a plasma that is
created by a corona-discharge at the end of a metal needle. APCI is
limited to forming singly-charged ions. The ionized molecules
formed with either source will then be analyzed by either a
quadrupole ion trap or a triple quadrupole mass spectrometer. The
experimental set-up using ESI source and a Waters TQD triple
quadrupole mass spectrometer is shown schematically in FIG. 6.
[0058] In order to create chemical images, software was developed
to allow for point sampling as well as continuous line scanning.
Point sampling allows for maximum extraction of a sample from a
given micrometer sized area as well as allow for the sampling of
material at any point on a surface. Sequential line scanning can be
used to create chemical distribution images from a sample
surface.
[0059] in order to understand the true quantity of the analytes
being tested to achieve nanometer scale resolution calculations
were performed. As will be understood, the longer the desorption
probe heats a certain region of a surface the larger the resulting
desorption crater. The heat transfer between the two bodies can be
modeled using Eq. 1 to estimate the diameter of craters that will
be created from different sized heating probes as a function of
time. The equation for heat transfer has the following form,
Q t = .kappa. A ( T hot - T cold ) d Eqn . 1 ##EQU00001##
where dQ is the heat transferred, dt is the change in time, .kappa.
is the thermal conductivity constant of the barrier, A is the area,
T is the temperature, and d is the thickness of the barrier.
[0060] FIG. 7 is a plot of crater size versus time, which
demonstrates that there is a linear dependence between the size of
a desorption crater and the time spent heating the surface. In
addition, the diameter of the desorption crater is strongly
dependent on the size of the heating probe. Therefore, it is
necessary to estimate the minimal size of a desorption crater that
will generate enough molecules to be detectable.
[0061] The number of molecules desorbed from a given desorption
crater using equation 2,
Moles = C * V * ( A 2 A 1 ) Eqn . 2 ##EQU00002##
where C is the concentration of a given sample, V is the total
amount of material, A.sub.1 is the total area of sample, and is the
desorbed area of the sample.
[0062] FIG. 8 is a graph of the number of moles desorbed from a
crater versus the concentration of the molecules on the surface for
several crater diameters generated using Equation 2. The curves
were calculated using a constant sample area (A.sub.1=1 cm.sup.2)
with A.sub.2 was set to the size of crater formed by the heated
probe. These results demonstrate that fmol amounts of molecules
will desorbed from a 250 micrometer sized crater. Smaller craters
(200 nm-2 .mu.m) reduce these amounts into the attomol range.
Example 2
[0063] FIG. 6 shows an experimental set-up for the thermal
desorption process described herein. Once the material is desorbed
by the thermally active tip 24 the gaseous analyte 42 will be
transferred from the desorption area 36 around the heated probe to
the ionization source 50 using a pump that creates a flow of gas
from the collection device inlet 44 probe into the ionization
source 50. The gaseous analyte 42 will then be ionized by the
ionization source (ESI or ANA) best suited for the targeted small
molecule being investigated.
[0064] FIG. 9(a) shows an AFM image of a specimen (S) with a
caffeine coating that was analyzed using the thermal desorption
technique described herein. The sampling was conducted by
positioning the thermally active tip a distance of 10 .mu.m above
the individual target site and heating the thermally active tip to
350.degree. C. The tip was then slowly lowered into contact with
the target site and maintained at the surface for 30 seconds at
which point the tip was cooled and removed from the surface. The
tip was them articulated so that it was positioned 10 .mu.m above
the next target and the process was repeated. In all there was a 20
second delay between when one thermal desorption cycle ended at one
target site and the next began at the adjacent target site. The
image of the specimen clearly shows the craters 58 formed at the
target sites by the thermal desorption technique.
[0065] FIG. 9(b) shows a plot of the topography of the surface of
the specimen shown in FIG. 9(a) taken along one of the sampling
lines. The plot shows that the craters are approximately 250 nm
wide and approximately 60-80 nm deep.
[0066] FIG. 9(c) shows the mass spectrometry results from the
thermal desorption process described with respect to FIG. 9(a). The
results are a relative intensity chronogram for SRM of caffeine
(m/z 195.fwdarw.138). Based on the calculations set forth above, it
is estimated that each thermal desorption evolved 6 attomol or 1.2
fg of caffeine. This example demonstrates the ability to obtain
sub-micron resolution chemical composition data.
Example 3
[0067] This Example focuses on an analysis where the specimen is a
high-performance thin-liquid chromatography (HPTLC) plate.
Analyte Chemicals
[0068] HPLC grade acetonitrile was purchased from Burdick and
Jackson (Muskegon, Mich., USA). HPLC grade methanol, ACS grade
chloroform, toluene and methylene chloride were obtained from J. T.
Baker, Inc, (Phillipsburg, N.J., USA). ACS grade acetone and ethyl
acetate were acquired from EM Sciences (Gibbstown, N.J., USA).
Glacial acetic acid, 99% ethanol, Sudan red 7B (CAS No, 6368-72-5),
2-acetoxybenzoic acid (aspirin, CAS No, 50-78-2) and
N-(4-hydroxyphenyl)ethanamide (acetaminophen or paracetamol, CAS
No. 103-90-2) were purchased from Sigma Aldrich (Milwaukee, Wis.,
USA), A test Dye Mixture V containing Sudan red 713, solvent green
3 (CAS No. 128-20-3), and solvent blue 35 (CAS No. 17354-14-2) in
toluene was obtained from Analtech, Inc, (P/N 30-05, Newark, Del.,
USA). Standard solutions (1000 mg/mL in acetonitrile) of
1,3,5,7-tetranitro-1,3,5,7-tetrazocane (UNIX, CAS No. 2691-41-0),
1,3,5-trinitroperhydro-1,3,5-triazine (RDX, CAS No. 121-82-4) and
2,4,6-trinitrotoluene (TNT, CAS No. 1.21-14-2) were obtained from
Supelco (Bellefonte, Pa., USA). Stock solutions (1000 mg/mL) of
1,3,5-triazine-2,4,6-triamine (melamine, CAS No. 108-78-1) in
diethylamine/water (80:20, v/v) were purchased from Restek (Restek
Corp., Bellefonte, Pa., USA), 2,4-Dichlorophenoxyacetic acid
(2,4-D, CAS No. 94-75-7), 4-(2,4-dichlorophenoxy) butyric acid
(4-(2,4-DB), CAS No. 94-82-6) and 2,4,5-trichlorophenoxyacetic acid
(2,4,5-T, CAS No, 93-76-5) were obtained from PolySciences Corp.
(Niles, IL, USA). 1,3,7-Trimethyl-1H-purine-2,6(3H,7H)-dione
(caffeine, CAS No. 58-08-2) was purchased from J. T. Baker. Extra
Strength Excedrin (Bristol-Meyers Squibb, New York, N.Y., USA)
containing 250 mg aspirin, 250 mg acetaminophen, and 65 mg caffeine
per tablet was purchased over the counter locally.
[0069] A solution of Sudan red 7B was prepared in methanol (2.4 mM)
for TLC and ESI/APCI-MS detection optimization. Standard solutions
from 0.01-10000 mg/mL of this dyestuff for TLC were prepared by
serial dilution of a methanolic stock solution with methanol.
Solutions of HMX, RDX, and TNT for EST/APCI-MS detection
optimization (4.4 mM) were prepared by dilution of 1000 mg/mL stock
solutions in acetonitrile. The analytical standards for TLC were
prepared by diluting this standard stock solution in acetonitrile.
Solutions of acetaminophen (1 mM), aspirin (1 mM), caffeine (1 mM),
4-(2,4-DB) (1.5 mM), 2,4-D (1.2 mM), and 2,4,5-T (1.5 mM) were
prepared for ESI/APCI-MS detection optimization by dissolving the
compounds in methanol.
[0070] In all cases, application of sample solutions to the TLC
plates was performed manually using a 10 mL syringe. Vertical
development of all the plates was carried out in a covered
flat-bottomed chamber.
[0071] The components, Sudan red 7B, solvent green 3, and solvent
blue 35, of Test Dye Mixture V (about 1000 .mu.g/mL of each
dyestuff), were separated on glass-backed normal phase silica gel
plates with organic binder and UV 254 indicator (HPTLC-HLF, 150
.mu.m phase, P/N 59077, AnalTech Inc., Newark, Del., USA). The dye
mix was spotted as a band (a closely spaced series of 1 .mu.L
aliquots) and the plate was developed in toluene. The developed
plates were dried in an oven at 110.degree. C. for 5 min just prior
to analysis. Photographs of the developed plates prior to analysis
were taken with an Olympus SP-500UZ digital camera (Olympus Imaging
Corp., Tokyo, Japan) using white light illumination. Serial
dilutions of Sudan red 713 (0.01-10 000 .mu.g/mL) in methanol were
prepared and 1 .mu.L aliquots with 5 mm spacing were applied to the
same type of HPTLC plate. The plates were developed in toluene and
then dried in an oven (110.degree. C.) for 5 min prior to
analysis.
[0072] The explosives TNT, RDX and HMX were separated on
glass-backed normal-phase HPTLC plates (ProteoChrom HPTLC silica
gel 60 F254, 150-200 .mu.m phase, P/N 1.05650.001, Merck KGaA,
Darmstadt, Germany) using a procedure adapted from Douse. The
explosives mix (1000 .mu.g/mL of each explosive) was spotted as a
band (a closely spaced series of 1 .mu.L aliquots). Serial
dilutions of TNT (0.001-1.000 .mu.g/mL) were prepared in
acetonitrile and 1 .mu.L aliquots with 5 mm spacing were applied to
the same type of HPTLC plate. A 1000 .mu.g/mL solution of TNT was
prepared in acetonitrile and 1 .mu.L aliquots with 5 mm spacing
were applied to the ProteoChrom HPTLC silica gel 60 plates,
glass-backed RP-18 HPTLC plates (HPTLC gel 60 RP-18 F254s, 150-200
.mu.m phase, P/N 13724/5, Merck KGaA, Darmstadt, Germany) and plain
glass slides (Gold Seal Products, Portsmouth, N.H., USA). The HPTLC
plates were developed in (v/v) chloroform/acetone and dried in an
oven (110.degree. C.) for 5 min prior to analysis. Photographs of
the developed plates prior to analysis were taken with an Olympus
SP-500UZ digital camera using short-wavelength UV illumination.
[0073] The TLC separation of herbicides, 4-(2,4-DB), 2,4-D, and
2,4,5-T, was performed using glassbacked normal-phase silica gel
plates with organic binder and UV-254 indicator (HPTLC-HLF, 150
.mu.m phase, P/N 59077, AnalTech Inc.). The herbicide mix (10000
mg/mL of each herbicide) was spotted as a band (a closely spaced
series of 1 .mu.L aliquots) and the plate developed in 85:15 (v/v)
toluene/glacial acetic acid. The developed plates were dried in an
oven (110.degree. C.) for 5 min prior to analysis. Photographs of
the developed plates prior to analysis were taken with an Olympus
SP-500UZ digital camera using short-wavelength UV illumination.
[0074] The separation of aspirin, acetaminophen, and caffeine
extracted from Excedrin tablets was carried out using a procedure
adapted from Williamson on glass-backed normal-phase silica gel
plates with organic binder and UV 254 indicator (HPTLC-HLF, 150
.mu.m phase, P/N 59077, AnalTech Inc.). The pharmaceutical
components were extracted from a ground-up fraction of an Excedrin
tablet using 50:50 (v/v) ethanol/ethyl acetate (1.4 mg of tablet/mL
of solution). The extract was centrifuged and filtered. The plates
were pre-developed with 99:1 (v/v) ethyl acetate/glacial acetic
acid and then dried in an oven at 110.degree. C. for 30 min. The
filtered solution containing aspirin, acetaminophen, and caffeine
was spotted as a band (a closely spaced series of 1 .mu.L aliquots)
and the plate was developed in 99:1 (v/v) ethyl acetate/glacial
acetic acid. The developed plates were dried in an oven
(110.degree. C.) for 5 min. Serial dilutions of acetaminophen
(0.001-1000 .mu.g/mL) were prepared in methanol and 1 .mu.L
aliquots with 5 mm spacing were applied to the same type of HPTLC
plate. The plates were developed in 99:1 (v/v) ethyl
acetate/glacial acetic acid and then dried in an oven (110.degree.
C.) for 5 mM prior to analysis. Photographs of the developed plates
prior to analysis were taken with an Olympus SP-500UZ digital
camera using short-wavelength UV illumination.
[0075] Aliquots (1 .mu.L) of a 1000 .mu.g/mL solution of melamine
in diethylamine/water (20:80) (v/v) were applied with 5 mm spacing
to glass-backed normal-phase HPTLC plates (ProteoChrom HPTLC silica
gel 60 F254), glass backed RP-18 HPTLC plates (HPTLC silica gel 60
RP-18 F254s, 150-200 .mu.m phase, P/N 13724/5, Merck KGaA), and
plain glass slides (Gold Seal Products). Both types of plates were
developed in 6:2:2 (v/v/v) acetonitrile/water/ethyl acetate and
then dried in an oven (110.degree. C.) for 5 min prior to
analysis.
TLC/Proximal Probe TD-MS System
[0076] FIG. 10 shows a schematic and a photograph of the
TLC/TD/I-MS experimental setup. The mass spectrometer used was a
Waters TQD triple quadrupole with ESCi capability (Waters Corp.,
Milford, Mass., USA). Analyte detection was performed using full
scan mode, single ion monitoring (SIM), or selected reaction
monitoring (SRM) with Ar as a collision gas (0.20 mL/min). The SRM
transitions monitored were: TNT (m/z 227.fwdarw.m/z 210, CE=10 eV),
Sudan red 7B (m/z 380.fwdarw.m/z 183, CE=16 eV), solvent blue 35
(m/z 351.fwdarw.m/z 251, CE=30 eV), solvent green 3 (m/z
419.fwdarw.m/z 327, CE=33 eV), acetaminophen (m/z 152.fwdarw.m/z
110, CE=16 eV) and melamine (m/z 127.fwdarw.m/z 85, CE=19 eV).
[0077] A special cone electrode in the TQD ionization source can
was fabricated by removing the normal curved gas inlet connection
and attaching a straight 1.5'' long stainless steel tube (1/8''
o.d., 1/16'' i.d.). This tube was connected to a modified Cajon
connector secured into the window of the ion source can. The normal
glass window in the door into the source can was replaced with
plexiglass. An opening for the Cajon connector was constructed from
two plexiglass pieces secured to one another with an O-ring and six
screws. Either a 12 V, 1.5 amp KNF N815KTE mini vacuum pump (KNF
Neuberger Inc., Freiburg, Germany), powered with a variable DC
supply, or a model MZ 2D vacuum pump (Vacuubrand GMBHCo, Werhteim,
Germany) with a F200S bleed valve (Parker Inc., Elyria, Ohio, USA)
was put in the ion source exhaust line. Pumping on the source
exhaust pulled air from the sampling region external to the source
block through the modified source can window and modified cone
electrode and into the ionization source. A model GPM 37 gas flow
meter (Aalborg Instruments, Orangeburg, N.Y., USA) was connected to
this gas inlet to calibrate gas flow rate into the source through
the cone connection.
[0078] An MD 80 wand from a WD 1 soldering station (Weller,
Germany) was used as the heated proximal probe. The exchangeable
heated probe tip used had a width of 1.6 mm and a thickness of 0.7
mm. A digital controller was used to adjust the temperature at the
probe tip from about 25 to 350.degree. C. The heated probe used was
mounted directly in front of the intake orifice into the ionization
region though the modified source can window and gas cone
electrode. The TLC plate was mounted so that the edge of the plate
was as close as possible to the sampling inlet used to draw gas and
vapors into the cone electrode region of the ionization source
block. The glass-backed TLC plates were cut along the length of or
perpendicular to the development lanes using a SmartCut device
(CAMAG, Wilmington, N.C., USA) to enable close positioning of the
heated probe and bands on the plate to the inlet region into the
ionization source. The plates were affixed to a platform on top of
the stage using double-sided tape.
[0079] The MS2000 x-y-z robotic platform (Applied Scientific
Instrumentation Inc., Eugene, Oreg., USA), and control software
used to manipulate the sample stage supporting the TLC plate
relative to the stationary heated probe. The stage could be moved
in all directions by manual or computer control to allow for
scanning of the developed TLC lanes. The initial positioning of the
stage and the sample to be investigated was done manually. The
development lane along the TLC plate was scanned in the x-y plane
under computer control. The exact position of the heated probe
relative to the surface during an experiment was monitored using a
CCD camera and a monitor.
Results
[0080] The schematic in FIG. 10 shows a setup for the following
TLC/TD/I-MS experiments. This TD/I system used a heated metal probe
placed close enough to just touch the surface of interest, but yet
not physically disrupt the surface during a scan of the surface
versus the stationary probe. Components desorbed from the surface
were drawn into the ionization region of the existing ESI/APCI
source, through the cone electrode, where they merged with reagent
ions and/or charged droplets from a corona discharge or an
electrospray emitter and were ionized. The ionized components were
then drawn through the atmospheric sampling orifice into the vacuum
region of the mass spectrometer and analyzed (FIG. 10).
[0081] Determining the position of the heated probe relative to the
inlet of the mass spectrometer was important for optimum
performance of the TLC/TD/I-MS system. This was accomplished by
mounting the probe on an x-y-z translation stage and adjusting the
vertical and horizontal position of the probe relative to the inlet
into the cone electrode while monitoring the signal intensity of
the analyte with the mass spectrometer. The region of the surface
to be analyzed was positioned as near to the sampling tube as
possible, within the constraints of the current instrument
interface design, and at the vertical mid-point of the inlet tube.
For the analysis of the TLC plates, this required the development
lanes to be near the edge of the plate. Therefore, the developed
plates were scored and cut parallel or perpendicular to the
development direction to provide access to the bands of interest
for analysis. Or course, alternate probe/inlet arrangements that
might include an extension on the inlet tube can be implemented to
allow analysis of uncut plates.
[0082] Beyond positioning of the heated probe and sample, the
performance of the TLC/TD/I-MS system was also dependent the
temperature of the heated probe, gas flow rate into the ionization
region, and surface scan speed relative to the stationary heated
probe. Optimal settings for the temperature of the heated probe
were investigated using the relatively volatile TNT. TNT was
applied to HPTLC plates as 1 .mu.g spots (1 .mu.L of a 1 mg/min
standard) with 5 mm spacing between the spots and then the plate
was developed. The TLC plate was scanned relative to the stationary
heated probe at a rate of 200 .mu.m/s while monitoring the signal
for the SRM transition of TNT. Multiple experiments were performed
using probe temperatures ranging from 100 to 350.degree. C. and a
gas flow rate into the ion source region of 30 mL/min (FIG. 11(a)).
A probe temperature of 350.degree. C., the highest temperature
possible with the current apparatus, produced the maximum signal
levels and was used for all subsequent studies. However, an even
higher probe temperature might further improve the signal levels
for this or other analytes, especially those less volatile than
TNT. However, thermal degradation of the surface or the compounds
of interest may become a factor at even higher temperatures.
[0083] Using this same TNT HPTLC separation, surface scanning, and
mass spectrometry detection protocol, the effect of gas flow rate
into the on source region on signal intensity was also
investigated. By varying the voltage applied to the KNF pump on the
ion source block, the gas flow rate into this region was varied
from 5.5 mL/min up to 30 mL/min. The gas flow rate from 40 mL/min
up to 62 mL/min was achieved using the Vacuubrand pump, by varying
a Parker bleed valve (FIG. 11(b)). The maximum signal levels were
achieved in the flow rate range of 30-40 mL/min. Note that this
flow rate was well within the recommended cone gas flow setting for
the mass spectrometer (viz. 0-833 mL/min). For simplicity of
operation; the KNF pump providing a flow rate of 30 mL/min into the
source region was used for all subsequent studies.
[0084] Effects of surface scan speed on analyte signal intensity
were also investigated using the Test Dye Mixture V. The mixture
was spotted (.about.1 .mu.g spots with 5 mm spacing) and developed
on an HPTLC plate. The peak height and area, and baseline peak
width measured for surface surface scan speeds of 50 to 800
.mu.m/s, with specific reference to the data obtained for the
separated spots of Sudan red 7B, are shown in FIG. 12. The largest
integrated peak area (SRM mode) was observed at the slowest scan
speed (50 .mu.m/s), with the peak area decreasing as the scan rate
increased (FIG. 12(b)). The observed peak height reached a maximum
plateau in the surface scan range from about 200-400 .mu.m/s (FIG.
12(c)). Importantly, the observed mass spectral chronographic band
widths (i.e., the time it takes to scan over a band) decreased with
scan speed and correlated well with the predicted mass spectral
band width values up to about 700 .mu.m/s (FIG. 12(a)). The
predicted mass spectral peak width, W, was calculated using Eqn.
(3), where dB is the diameter of the analyte band determined
visually, dP is the diameter of the probe, and r is the scan speed.
The diameter of the probe was included Eqn. (3) to account for the
TD of the analyte from the time that the near side of the probe
first probe approaches the band until the far side of the probe
passes completely through the band.
W = ( d B + d P ) r Eqn . ( 3 ) ##EQU00003##
[0085] At the fastest scan speeds (.about.700 to 800 .mu.m/s) the
measured peak width reached a plateau and then began to diverge
from the predicted peaks widths. This trend was attributed to the
finite time necessary for the vapors from the desorbed species to
pass into and through the interface and be detected by the
instrument. The same set of experiments was carried out with the
other two dyestuffs from the mixture, viz., solvent green 3,
solvent blue 35, as well as with TNT. The same basic trends in peak
height and peak width were also observed for these compounds.
[0086] The detection levels for this TLC/TD/I-MS technique were
examined for three different compounds of greatly differing
volatility, viz., TNT, acetaminophen and Sudan red 7B, listed in
order of decreasing volatility. Aliquots (1 .mu.L) of serial
dilutions of TNT (0.001-1000 .mu.g/mL), acetaminophen (0.001-1000
.mu.g/mL), and Sudan red 7B (0.01-10000 .mu.g/mL) were spotted on
the appropriate HPTLC plates with 5 mm spacing and the plates
developed as described in the Experimental section. The SRM
transitions for these compounds were monitored in positive ion mode
APCI while scanning the development lanes at 200 .mu.m/s. The
normalized peak areas measured versus plate loading are shown in
FIG. 14.
[0087] These calibration data were evaluated using a least squares
regression and fit the model A-bx+a, where A is the integrated peak
area for a compound with mass x spotted on the TLC plate. The
values b and a are the slope and intercept, respectively, of the
calibration curve, and are presented for each compound in Table 1.
From the linear calibration curves the detection limit was
estimated (3.sub.x/y/slope, where s.sub.x/y, the standard error of
the y value estimates, is assumed to approximate the standard
deviation of the blank, s.sub.B). This translates to detection
levels of 24 ng (0.11 nmol), 370 ng (2.4 nmol), and 5700 ng (15
nmol) for TNT, acetaminophen and Sudan red 7B, respectively (Table
1, below). Thus the best detection level was obtained for the more
volatile compound. This might be expected to be a general trend for
a TD-based process, but other factors such as varying ionization
efficiency among compound types will also affect detection levels.
While these detection levels are not exceptional from a mass
spectrometric point of view, they are within typical plate loadings
for HPTLC (i.e., low .mu.g levels), especially for the more
volatile compounds.
TABLE-US-00001 TABLE 1 Figures of merit for the calibration curves
and calculation of the detection limit for TLC/TD/I-MS Compound TNT
Acetaminophen Sudan red 7B Calibration range, ng 1-100 100-1000
125-10000 Slope (b) .+-. Std dev 2.17 .+-. 0.11 0.027 .+-. 0.003
1065 .+-. 87 Intercept (a) .+-. Std dev 1.0 .+-. 5.7 5.8 .+-. 1.6
-212 .+-. 430 r.sup.2 0.95 0.89 0.78 Standard error of the y 17 3.3
2015 value estimates, S.sub.x/y or S.sub.B Detection limit, ng (3 *
24 370 5700 S.sub.B/b) Molecular weight, ng/n 227 152 379 mol
Detection limit, nmol 0.11 2.4 15
[0088] It is worth comparing how the signal levels vary for any one
analyte when desorbed from different stationary phases and simply
from a plain glass surface. This surface effect was illustrated for
both melamine and TNT from glass-backed ProteoChrom HPTLC plates
and glass-backed RP-18 HPTLC plates as well as from plain glass
slides. Solutions of TNT (1000 .mu.g/mL) and melamine (1000
.mu.g/mL) were spotted in 1 .mu.L, aliquots with 5 mm spacing on
the respective plates and the HPTLC plates developed. The SRM
transitions of TNT and melamine were monitored in positive ion mode
APCI while scanning the development lanes (100 .mu.m/s for TNT and
300 .mu.m/s for melamine) relative to the heated probe. The largest
in rated peak area for both melamine and TNT was observed when
scanning across the analyte spots on plain glass slides and lowest
when analyzing the developed spots on the NP plates. The different
surfaces had a similar effect on the signal levels for both TNT and
melamine. With both these relatively polar compounds, the signal
from the plain glass surface was .about.10 times greater than that
from the NP plate and .about.5 times greater than the signal level
from the RP-18 plate. Obviously, the nature of the surface and
particular analyte/surface interactions will influence the signal
levels and limits of detection.
Selected Applications
[0089] Beyond the investigation of variable parameters and
performance metrics discussed above, four mixtures of significantly
different analyte types, viz., pharmaceuticals, solvent dyestuffs,
herbicides and explosives, were examined to illustrate the
prospective applicability of this proximal probe TD/I approach for
coupling TLC and MS. The pharmaceutical application is described
below in more detail.
[0090] An Extra Strength Excedrin tablet containing 65 mg of
caffeine, 250 mg of acetaminophen, and 250 mg of aspirin was
ground-up and the extract containing these three compounds was
spotted as a series of tightly spaced spots containing 1.4 .mu.g of
material per spot and separated on a glass-backed NP HPTLC-HLF
silica gel plate. FIG. 14(a) shows a picture of the development
lane and the three separated bands. To acquire the mass spectral
data shown in FIGS. 14(b)-14(e), the HPTLC plate was scanned (200
.mu.m/s) from low to high RF (left to right in the picture)
relative to the heated probe (350.degree. C.) while full scan mass
spectral data was acquired. In this case, two of the compounds,
caffeine and acetaminophen, were most readily ionized and detected
as the respective protonated molecules using positive ion mode
APCI. The detection of aspirin was optimized by using ESI to form
the sodiated adduct [M+Na].sup.+. That being the case, positive ion
mode ESCi was used for this experiment with the ion source
switching scan to scan between APCI and ESI mode. To promote ESI,
methanol was sprayed through the ESI probe at a flow rate of 60
.mu.L/min. The averaged, background-subtracted mass spectra
obtained while scanning the respective bands are shown as insets in
FIGS. 14(c)-14(e) for caffeine ([M+Na].sup.+, m/z 195),
acetaminophen ([M+Na].sup.+, m/z 152), and aspirin ([M+Na].sup.+,
m/z 203), respectively. This group of compounds in particular
highlights the versatility of the technique by utilizing the APCI
and ESI sources in tandem for the detection of analytes with
varying ionization requirements.
[0091] Using this set of data, the chromatographic resolutions
obtained by HPTLC and MS were also compared. The chromatographic
resolution, R, of two chromatographic bands was calculated using
Eqn. (4), where d is the distance between the centers of the bands,
and W1 and W2 are the widths of the two bands.
R = d ( W 1 + W 2 ) / 2 Eqn . ( 4 ) ##EQU00004##
From the optical data obtained using the photograph of the plate in
FIG. 15(a), the chromatographic resolution for the TLC separation
of caffeine (C) and acetaminophen (A) was calculated as
(R.sub.C/A)=3.64 and for acetaminophen (A) and aspirin (S) as
(R.sub.A/S)=1.54. From the mass spectral data we calculated
(R.sub.C/A)=3.42 and (R.sub.A/S)=1.38, or about 6-10% lower than
the apparent chromatographic resolution. This was considered to
represent good agreement between the two data sets (optical and MS)
given the difficulty in accurately determining the extent of the
bands on the plate by simple visual observation.
[0092] FIG. 14(a) is a black and white photograph of glass-backed
normal-phase silica gel plate (HPTLC-HLF) development lane showing
the separated bands of a three-component Excedrin mixture
containing caffeine, acetaminophen and aspirin. FIG. 14(b) shows
the total ion current from full scan ESCi mode, while FIG. 14(c),
(d) and (e) show individual extracted ion current chromatograms for
(c) caffeine (m/z 195) using APCI, (d) acetaminophen (m/z 152)
using APCI, and (e) aspirin (m/z 203) using ESL respectively. Also
shown in panels for FIGS. 14(c)-(e) are the averaged,
background-subtracted full scan mass spectra (m/z 115-215) for the
respective compounds. The development lane was scanned at 200 mm/s
relative to the heated probe (350.degree. C.). The solution
containing caffeine, acetaminophen and aspirin was spotted as a
band (a closely spaced series of 1.4 mg loadings) on the HPTLC
plate.
[0093] The data demonstrate that a simple proximal probe thermal
desorption/ionization approach for coupling TLC and MS. The
experimental setup was optimized for probe and plate positioning
relative to the inlet into the ionization region of the mass
spectrometer as well for the variable parameters of probe
temperature, gas flow into the mass spectrometer and surface scan
speed using single lane scans for selected analytes developed on
various HPTLC plates. The experiments showed that the compound band
widths determined by mass spectrometry matched the chromatographic
band width up to surface scan speeds of about 700 mm/s.
[0094] The wide ranging applicability of this TLC/TD/I-MS technique
was demonstrated using compounds with very disparate volatilities
and ionization behavior, including dyestuffs, herbicides,
explosives and pharmaceuticals. The use of a commercial ionization
source capable of operation in ESI, APCI or ESCi mode added to the
usefulness of the present approach. The ESCi approach might be
expected to be particularly useful in a discovery mode where the
nature of the analytes and the best ionization method to utilize
may not be known.
[0095] While the invention has been described in terms of specific
embodiments, it is evident in view of the foregoing description
that numerous alternatives, modifications and variations will be
apparent to those skilled in the art. Accordingly, the invention is
intended to encompass all such alternatives, modifications and
variations which fall within the scope and spirit of the invention
and the following claims.
* * * * *